The roles of TGF-β and VEGF pathways in the suppression of antitumor immunity in melanoma and other solid tumors

Immune checkpoint blockade (ICB) has become well-known in cancer therapy, strengthening the body’s antitumor immune response rather than directly targeting cancer cells. Therapies targeting immune inhibitory checkpoints, such as PD-1, PD-L1, and CTLA-4, have resulted in impressive clinical responses across different types of solid tumors. However, as with other types of cancer treatments, ICB-based immunotherapy is hampered by both innate and acquired drug resistance. We previously reported the enrichment of gene signatures associated with wound healing, epithelial-to-mesenchymal, and angiogenesis processes in the tumors of patients with innate resistance to PD-1 checkpoint antibody therapy; we termed these the Innate Anti-PD-1 Resistance Signatures (IPRES). The TGF-β and VEGFA pathways emerge as the dominant drivers of IPRES-associated processes. Here, we review these pathways’ functions, their roles in immunosuppression, and the currently available therapies that target them. We also discuss recent developments in the targeting of TGF-β using a specific antibody class termed trap antibody. The application of trap antibodies opens the promise of localized targeting of the TGF-β and VEGFA pathways within the tumor microenvironment. Such specificity may offer an enhanced therapeutic window that enables suppression of the IPRES processes in the tumor microenvironment while sparing the normal homeostatic functions of TGF-β and VEGFA in healthy tissues.


Introduction
Since a healthy immune system is innately able to suppress tumors, it has been suggested that cancer is synonymous to immune dysfunction (Zappasodi, Merghoub, & Wolchok, 2018).Thus, reinvigorating tumor-specific immune response is a promising way to control and cure cancer.The remarkable clinical results of blocking immune inhibitory checkpoints such as programmed cell death protein (PD-1), programmed cell death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte antigen-4 (CTLA-4) in some cancers during the last decade have propelled immune checkpoint blockade (ICB)-based immunotherapy into popularity.Approximately one third of patients with advanced metastatic melanoma responded to ICB using monoclonal antibody (mAb) against PD-1 (anti-PD-1) (Hamid et al., 2013;Robert et al., 2015Robert et al., , 2015;;Weber et al., 2015).Subsequently, the combination of anti-PD-1 and anti-CTLA-4 was approved as a first line therapy for the treatment of patients with unresectable or metastatic melanoma (Larkin et al., 2015;Postow et al., 2015).The combination therapy regimen achieved an objective response rate of 59%, but it was accompanied by a high frequency of grade 3/4 treatment-related adverse events (trAEs) caused by a hyperactivated immune system (Johnson, Nebhan, Moslehi, & Balko, 2022;Larkin et al., 2015;Postow et al., 2015).
In line with the role of the TGF-β pathway in dampening antitumor immune response, Jiang et al. showed that TGF-β1 transcript levels are significantly correlated with T cell dysfunction only in melanoma displaying high cytotoxic T cell (CTL) infiltration (Jiang et al., 2018).This report also highlighted a general anti-correlation between the levels of CTL and immunosuppressive immune populations such as M2-like, tumor-associated macrophages (TAMs), MDSCs, and CAFs.In microsatellite stable colorectal cancer (MSS CRC), both wound healing signatures and VEGFA mRNA expression correlated with later disease stage (Kim et al., 2019).Instead of wound healing signatures, the microsatellite instability-high colorectal tumors (MSI CRC) are enriched with interferon gamma (IFN-γ)related gene signatures; higher IFN-γ and lower wound healing signature enrichments were proposed to be the drivers of the ICB response in MSI CRC but not MSS CRC.
Other studies also reported the correlation between the enrichment of stromal signatures and worsened prognosis in patients with melanoma, gastric, metastatic urothelial and colorectal cancer (Calon et al., 2015;Zeng et al., 2019Zeng et al., , 2021)).Zeng et al. devised a combined score of the TME (termed "TMEscore"), which considers the immune-and stromal-activation scores, to predict the overall survival of gastric cancer patients.Notably, TMEscore can predict response in ICB-treated melanoma, metastatic urothelial carcinoma (Zeng et al., 2019) and metastatic gastric cancer (Zeng et al., 2021).In a separate study, high expression of CAF and TGF-β signaling marker genes identify patients with poor-prognoses across CRC subtypes (Calon et al., 2015).Using tumor organoid models of human CRC that express high level of TGF-β, abrogation of TGF-β signaling was shown to significantly reduce tumor metastasis in mice.
Thus, multiple analyses of large cancer datasets have demonstrated a significant (anti) correlation between activities of TGF-β and VEGFA pathways and levels of antitumor immune response.The next logical question is whether these pathways are readily targetable, and if so, whether targeting them can improve the efficacy of existing ICB therapies.
VEGFA, along with VEGFB and PlGF, also binds the VEGFR-1 receptor.Interestingly, while VEGFA binds more strongly to VEGFR-1 (Flt-1), the lack of independent mitogenic or angiogenic effect of the VEGFA-VEGFR-1 interaction suggests that VEGFR-1 may function as a negative regulator of VEGFR-2 activation (Park, Chen, Winer, Houck, & Ferrara, 1994).VEGFB signaling through VEGFR-1 does not have a direct effect on the proliferation and survival of endothelial cells but is required for the development of normal heart vasculature and recovery from heart ischemia (Bellomo et al., 2000).On the other hand, PlGF binding to VEGFR-1 can either directly induce angiogenic processes via Akt pathway activation or indirectly enhance the VEGFA-VEGFR-2 pathway by occupying VEGFR-1 (Autiero, Luttun, Tjwa, & Carmeliet, 2003;Fischer, Mazzone, Jonckx, & Carmeliet, 2008).PlGF pathway activation not only induces vascular development and maintenance in healthy tissues but also acts as an angiogenic switch in cancer (Fischer et al., 2008).The other VEGF proteins, VEGFC and VEGFD, are implicated in the regulation of lymphoangiogenesis through their specific binding to VEGFR-3 (Alitalo, Tammela, & Petrova, 2005;Karkkainen et al., 2004).
The FDA-approved VEGF-trap, aflibercept, is indicated for mCRC (Stewart, 2011).Of note, the combination of aflibercept and pembrolizumab displayed an acceptable safety profile with antitumor activity in a phase 1 study on patients with melanoma, RCC, and mesothelioma (Tyan et al., 2021).In general, the combination of VEGFA targeting and ICB has an acceptable safety profile that is comparable to that of the standard of care.As such, we expect more clinical trials testing the combination of VEGFA pathway inhibition and ICB in more diverse cancer types, especially those on which ICB alone is less efficacious.
The TGF-β ligand has three isoforms: TGF-β1, -β2, and -β3.Each starts as an inactive precursor protein containing a signal peptide, a latency-associated polypeptide (LAP), and the mature C-terminal polypeptide (Hinck, Mueller, & Springer, 2016;Morikawa et al., 2016;Moses, Roberts, & Derynck, 2016).Two precursor proteins subsequently dimerize through the formation of a disulfide bond across the mature polypeptide region.The Nterminal LAP is proteolytically cleaved by furin but stays non-covalently associated with the TGF-β dimer.This complex (termed the small latent complex) can associate through disulfide bonding with latent TGF-β binding protein (LTBP) into a large latent complex (LLC) that is bound to ECM proteins such as collagen, thrombospondin and fibronectin.The small latent complex can also bind glycoprotein-A repetitions predominant (GARP) proteins on the plasma membrane.These arrangements allow the deposition of TGF-β ligands that can only initiate the downstream signaling after an activation-driven cleavage from the ECM/LTBP (hence their "latent" characteristic) (Robertson & Rifkin, 2016).
Knockout mouse studies for the three TGF-β isoforms have been used to further elucidate their specific roles.TGF-β1 is important for hematopoiesis and vascular development (Dickson et al., 1995).Additionally, TGF-β1 expression and activation are rapidly upregulated in response to injury, and are crucial for efficient wound healing in vivo (Kane, Hebda, Mansbridge, & Hanawalt, 1991;Sporn et al., 1983).TGF-β2 contributes to development of the skeleton, heart, eyes, ears, and urogenital tract (Sanford et al., 1997).TGF-β3 is necessary for the development of the pulmonary system where a deficit leads to cleft palates and death (Proetzel et al., 1995).In addition, mice deficient in TGF-β2 and -β3 expression reveal defects in their central nervous system (Vogel, Ahrens, Büttner, & Krieglstein, 2010).

Trap antibodies: localized targeting of TGF-β
Given TGF-β's critical function in maintaining immune homeostasis (Horwitz, Fahmy, Piccirillo, & La Cava, 2019;Sanjabi, Oh, & Li, 2017), systemic targeting of the TGF-β pathway can result in serious adverse events such as cardiovascular inflammation (Colak & ten Dijke, 2017;Teixeira, ten Dijke, & Zhu, 2020).Thus, therapies targeting TGF-β need to be localized to the tumor site and/or specific cell populations associated with TGF-β ligands.An antibody-ligand "trap", a class of bispecific antibodies, can accomplish this localization goal (Ravi et al., 2018).The constant region of the bispecific antibody binds the target ligand, while the variable domains of the antibody bind to a specific cell surface marker; this antibody effectively "traps" the target ligand near the target cell.In short, we refer to this type of antibody as "trap antibody" (see Fig. 1).When many trap antibodies bind their target marker on cell surfaces, they can efficiently sequester the target ligands near target cells by virtue of their high local concentration.This mechanism of action results in a localized, cell type-specific reduction of the unbound ligand around and subsequent suppression of pathway activation by the ligand within the target cell population.
For instance, 4T-Trap is a trap antibody that traps TGF-β ligands while binding to CD4 receptors on T cells (Li et al., 2020).4T-Trap is engineered by adding TβRII's extracellular domain to the constant region of ibalizumab (a non-immunosuppressive CD4 antibody).4T-Trap was designed based on the observation that loss of TβRII in CD4 + T cells but not CD8+ T cells suppressed the growth of PyMT (a mouse model of breast cancer) and MC38 (colorectal cancer mouse model) (Liu et al., 2020).Specifically, Liu et al. reported that the antitumor effect of TβRII loss was mediated by enhanced Th2 differentiation and interleukin-4 (IL-4) cytokine expression by CD4+ T cells.The activation of Th2 T cells renormalized tumor vasculature, causing cancer cell hypoxia and death.Notably, in both models, antitumor response driven by TβRII loss is fully dependent on the Th2 cytokine, IL-4.
Li et al. utilized 4T-Trap to mimic the specific deletion of TβRII in CD4 + T cells (Li et al., 2020).When they compared 4T-Trap to a non-targeted TGF-β-trap, they observed that only 4T-Trap recapitulated the tumor vascular normalization and IL-4 induction in TβRIIdeficient CD4+ T cells.4T-Trap treatment subsequently induced hypoxia-driven tumor cell death in mice with PyMT and MC38 tumors.Of note, the authors suggested that one of the major sources of the TGF-β1 ligand were the activated CD4+ T cells themselves (i.e., autocrine TGF-β signaling).Thus, the efficacy of 4T-Trap may also be attributed to its ability to efficiently sequester (and internalize) TGF-β1 ligands as they are being secreted by activated CD4+ T cells (for an illustration of the mechanism of action of 4T-Trap, see Fig. 2).Furthermore, the tumor draining lymph nodes (tdLN) of 4T-Trap treated mice were enriched in effector memory CD4+ T cells, thereby demonstrating showing a suppressed TGF-β pathway activity.This observation suggests the ability of 4T-Trap to activate antitumor CD4+ T cells outside the TME.The induction of tumor hypoxia by 4T-Trap upregulated VEGFA expression, which motivated the authors' targeting of both the TGF-β and VEGFA pathways in PyMT and MC38 tumor models.Indeed, co-administration of 4T-Trap and a VEGF-trap (modeled after the human VEGF-trap, aflibercept) synergistically suppressed tumor growth and prolonged mice survival (Li et al., 2020).This result strongly supports the notion of dual targeting of TGF-β and VEGFA (i.e., simultaneous targeting of IPRES processes) to achieve stronger antitumor activity than targeting either pathway alone.
Besides 4T-Trap, other effective preclinical and clinical trap examples, primarily combining TGF-β targeting agents with ICB, have been reported in recent years.For instance, Ravi et al. showcased the superior antitumor efficacy of two TGF-β trap antibodies, which were engineered from FDA-approved antibodies targeting CTLA-4 (ipilimumab) or PD-L1 (atezolizumab and avelumab) immune checkpoints (Ravi et al., 2018).For brevity, we will refer to these trap antibodies as CTLA4-TβRII trap and PDL1-TβRII trap, respectively.Using melanoma and triple negative breast cancer (TNBC) human cancer cell lines xenografted into NSG mice that were immune reconstituted using HLA-matched human bone marrow cells, Ravi et al. reported enhanced antitumor activity of CTLA4-TβRII trap over anti-CTLA4 monotherapy, a non-specific TGF-β-trap, as well as their combination.Tumors from mice treated with CTLA4-TβRII trap displayed higher proportions of 1) tumor reactive CD8+ IFNγ+ T cells, 2) CD4+ and CD8+ central memory T cells, and 3) lower percentage of FOXP3+ Tregs compared to control mice.
Because CTLA-4 is constitutively highly expressed in Tregs, and given Tregs' dependence on the TGF-β pathway to maintain its activity (Chen et al., 2003;Tone et al., 2008), CTLA4-TβRII trap effectively prevented Treg differentiation and activity.CTLA4-TβRII trap also effectively suppressed the differentiation of CD4+ T helper cells to the Th17 lineage (inflammatory and autoimmune-related) since Th17 differentiation depends on IL-6 and TGF-β ligand.Strikingly, the authors observed that CTLA4-TβRII trap alone inhibits the growth of the TNBC tumor model better than a combination treatment using anti-CTLA-4 plus anti-PD1.The authors further reported the efficacy of PDL1-TβRII trap in suppressing tumor growth in the melanoma and TNBC models.The authors implicated that PDL1-TβRII functions by sequestering TGF-β near PD-L1-expressing tumor cells.As with CTLA4-TβRII, PDL1-TβRII reduced the proportion of intratumoral Tregs; the mechanism underlying this reduction was not described in detail.Since PD-L1 is not usually highly expressed on the surface of Tregs, it is possible that the localized sequestration of TGF-β in the TME indirectly limits the availability of unbound TGF-β ligand for Treg differentiation and activity.
Confirming the utility of sequestering TGF-β near PD-L1+ cell population, an independent study demonstrated the efficacy of M7824, a PDL1-TβRII trap (based on avelumab), in suppressing tumor growth and metastasis in orthotopic breast and colorectal cancer models (Lan et al., 2018).Importantly, M7824 conferred antitumor immunological memory that protected mice from tumor rechallenge long after treatment discontinuation.Combined treatment of M7824 with radiation therapy was shown to suppress the growth of not only the irradiated subcutaneous MC38 tumor but also the non-irradiated, opposite flank MC38 tumor.Such an abscopal effect, combined with a hint of immunological memory formation, strongly suggests that M7824 is capable of inducing a systemic, tumor-specific immune response.Unlike 4T-Trap, whose efficacy depends on CD4+ T cells, the authors showed that the antitumor activity of M7824 was dependent on cytotoxic CD8+ T cells and NK cells.In an in vitro study of M7824, Grenga et al. showed the ability of M7824 to modulate the immunogenicity of urothelial carcinoma cells, thus making them more susceptible to immune surveillance (Grenga et al., 2018).Specifically, the authors demonstrated that M7824 mediates NK cell-driven antibody-dependent cellular cytotoxicity against the tumor cells in vitro.Additionally, compared to anti-PD-L1 monotherapy, M7824 more strongly induced upregulation of intratumoral T-cell trafficking genes such as CXCL11 as well as bolstered antigen-specific cytotoxic T cell-mediated tumor cell lysis.
On the basis of favorable results from multiple preclinical studies, M7824 underwent a phase 1 clinical trial in a cohort of nineteen heavily pretreated patients with advanced solid tumors (Strauss et al., 2018).M7284 treatment led to one confirmed complete response in a patient with cervical cancer, near partial response in another patient with cervical cancer, and two durable confirmed partial responses in pancreatic and anal cancers.In two patients (with pancreatic cancer and carcinoid) who experienced progressing disease at the time of study entry, M7824 induced stable disease.Four of nineteen patients experienced grade three or higher adverse events such as skin infection secondary to localized bullous pemphigoid, anemia-associated colitis, and gastroparesis.Overall, M7824 seems to exhibit a manageable safety profile.Another phase I trial testing M7824 on patients with metastatic/locally advanced solid tumors in Asia (NCT02699515) also showed the clinical promise of M7824 (Bang et al., 2018).Combining the results of patients from original and expansion cohorts, 7 out of 31 heavily pretreated patients with advanced gastric cancer achieved an objective response (5 partial responses and 2 complete responses).Seven patients experienced grade 3-5 trAEs: anemia (2), diarrhea (1), abnormal hepatic function (1), rash (2) and 1 grade 5 AE (suspected rupture of pre-existing thoracic aortic aneurysm).
Despite the initial successes of M7824, it is important to note that several clinical studies were terminated early (see Table 2).One such example was a phase III study comparing the efficacy of M7824 as a first line treatment for patients with advanced, PD-L1 positive NSCLC.The comparator arm was pembrolizumab (the FDA-approved ICB for this cancer type).The interim analysis indicated that the trial was likely to miss its primary end point: progression free survival (PFS).We speculate that the TGF-β ligand's main source/target cell population(s) in the NSCLC TME are likely not in the vicinity of PD-L1 expressing cell populations.Hence, this patient population could not leverage the bispecific merit of M7824.Other studies were terminated due to serious trAEs and/or tumor hyperprogression (Table 2).TGF-β signaling blockade has been associated with increased risk of bleeding, presumably caused by compromised vascular integrity.After all, TGF-β signaling on the pericytes is required for endothelial integrity (Derynck et al., 2021).Instances of tumor hyperprogression are of serious concern.In an inflamed TME with high PD-L1 expression, immune cell-derived TGF-β1 may suppress tumor proliferation; localized TGF-β blockade by M7824 may negate such suppression.
Nonetheless, additional studies on the immune, stromal, and tumor cell populations from the treatment-responding and -non-responding tumors are needed to dissect the mechanism of action (and non-action) of M7824.Such knowledge will be crucial to improve the design of future TGF-β-traps and to stratify patient populations that can benefit most optimally from M7824.

Discussion
Undermining IPRES by blocking the activity of its key pathways, VEGFA and TGF-β, has robust potential to improve clinical outcomes of patients with melanoma treated with ICB.Systemic targeting of VEGFA, along with its combination with ICB, are generally well tolerated in patients.The most frequent trAEs were hypertension or proteinuria, which were also commonly observed upon anti-VEGFA monotherapy and are generally manageable.As such, the combination of anti-VEGFA and ICB is being tested in a multiple tumor histologies.Thus far, the benefit of combined VEGFA and immune checkpoint inhibition is seen in tumors that respond to single agent anti-VEGFA therapy such as HCC, RCC, CRC, NSCLC, and gynecologic tumors (Table 1).One exception is glioblastoma, where the combination of anti-PD-1 and anti-VEGFA was not better than administering anti-VEGFA as a single agent (Reardon et al., 2018).In melanoma, improvements in overall survival by a single agent targeting VEGFA have historically been limited (Corrie et al., 2018).Nevertheless, several ongoing clinical trials are testing the combination of anti-VEGFA and anti-PD-1/PD-L1 in metastatic melanoma (NCT02681549, NCT04356729, NCT03175432).
More considerations should be factored into the design of strategies targeting the more pleiotropic TGF-β signaling.The treatment dosage and regimens of existing TGFβ inhibitors, anti-TGFβ antibodies or M7824 often have a relatively narrow therapeutic window as it is common for potent systemic inhibition of TGF-β signaling to confer substantial toxicities (Derynck et al., 2021).M7824, a PDL1-TβRII trap that binds TGF-β from the sites with high expression of PD-L1, has shown potential clinical efficacy in a phase 1 basket clinical trial of multiple solid tumor types (Strauss et al., 2018).It is worthwhile to note that a few of the subsequent clinical trials of M7824 were terminated or withdrawn, again due to safety concerns.It is possible that systemic T cell activation induced by the anti-PD-L1 portion of M7824 can also induce PD-L1 expression in other organs beyond the local TME.In such cases, localized sequestration of the TGF-β ligand near the (inflamed) PD-L1+ normal tissue will prevent the normal homeostatic response against such inflammation and result in immune-mediated toxicities.Although 4T-Trap is still in a preclinical stage, its merit of localized TGF-β inhibition in a specific antitumor CD4+ T cell population may result in potent antitumor effects as well as a more manageable toxicity profile (Li et al., 2020).Of note, the authors demonstrated that the combination of 4T-Trap and VEGF-trap, which targets two IPRES pathways, resulted in significantly stronger tumor control in mice.Since the combination of VEGF-trap with anti-PD-1 was found to be safe in patients with cancer (Tyan et al., 2021), a potential future combination regimen may involve the co-administration of 4T-Trap with VEGF-trap and ICB.
Anti-VEGF therapies are associated with dose-limiting cardiovascular and non-cardiovascular toxicities despite their generally acceptable safety profiles (see http://www.uptodate.com/contents/toxicity-of-molecularly-targetedantiangiogenicagents-cardiovascular-effectsand http://www.uptodate.com/contents/toxicityof-molecularly-targeted-antiangiogenicagents-non-cardiovascular-effects).Thus, in the same vein as the design of 4T-Trap, a cell type-specific VEGF-trap may also hold potential to enhance ICB efficacy in melanoma and other solid cancers.Given the specific inhibitory effects of VEGFA on tumor-reactive, cytotoxic CD8+ T cells (Gavalas et al., 2012;Kim et al., 2019), a VEGFA-trap directed to activated CD8+ T cells may improve antitumor T cell activities in VEGFA-rich TME.Indeed, VEGFA can induce the activation of the master regulator of T cell exhaustion, TOX, as well as the expression of the PD-1 checkpoint in tumor-reactive, CD8+ T cells (Kim et al., 2019).These observations motivate the design of a PD-1 directed, VEGFA trap antibody that binds specifically to PD-1+ T cells and protects them from VEGFA mediated suppression (a schematic of how PD-1-VEGFA-trap may function is illustrated in Fig. 3).One such PD-1-VEGFA-trap, AK112 (a humanized IgG1 bispecific anti-PD-1/VEGFA antibody), is currently being tested in multiple phase 2 clinical trials involving NSCLC, TNBC, and advanced gynecological tumors (NCT04736823, NCT05227664, NCT04870177).Results from a phase 1b trial of AK112 on patients with advanced/metastatic solid tumors that are refractory to standard therapies revealed a favorable safety profile and provided preliminary evidence of antitumor activity (Coward et al., 2021).While adverse events did occur in 55.2% of the patients, only three out of 29 patients experienced grade 3 trAEs, and no grade ≥ 4 AEs occurred.Of the 17 patients treated at doses ≥3 mg/kg once every two weeks, the objective response rate (ORR) was 23.5% (4/17) and disease control rate (DCR) was 64.7% (11/17).Given the fact that the tumors were highly refractory to existing therapies, this result highlights the potency of AK112.It remains to be seen if the response rate holds in the later phases of AK112 clinical testing.
It is possible that the application of AK112 or other cell surface marker-specific VEGF-Trap can induce intracrine VEGFA signaling.In this mode of signaling, the VEGFA protein activates the VEGFR-1 or VEGFR-2 receptor from within the cell (e.g., in the endoplasmic reticulum or the nucleus) (Wiszniak & Schwarz, 2021).Upon binding to the PD-1 receptor of CD8+ T cells, AK112-trapped VEGFA could dissociate from it in the acidic environment of the endosome, bind to VEGFR-2 and activate VEGFA signaling in the target T cells in an intracrine manner.Such a process could negatively affect T cells.Additional studies are needed to ascertain if 1) AK112 is internalized after binding to PD-1 and, 2) there is any evidence of intracrine VEGFR-2 phosphorylation in the T cells with AK112 treatment.In the case where intracrine VEGFA signaling is present, instead of using an antibody against VEGFA, one can utilize aflibercept, a recombinant VEGFR mimic, in the design of the PD-1-VEGFA-trap.The significantly higher binding affinity between aflibercept and VEGFA compared to VEGFR-2 and VEGFA should diminish the possibility of VEGFA dissociation from the trap antibody and subsequent binding to VEGFR-2 after PD-1 receptor internalization.
The efficacy of ICB in various cancers has informed a robust discussion between clinicians, scientists, and pharmaceutical stakeholders on optimal dosing and therapeutic regimens to maximize response rate, minimize toxicity, and improve survival of cancer patients.The application of novel trap antibodies against the immunosuppressive pathways represented by IPRES may uncover novel synergistic combinations with existing ICB-based immunotherapies.Such combinatorial treatments could optimally harness the immune system to suppress and eventually eradicate tumors in patients with cancer.The general structure of a trap antibody.Schematic of a generic trap antibody structure.The variable regions/F ab (shown in gray) are specific for a cell-surface protein marker on a target cell population.The constant region/F c (shown in brown) is fused to either an antibody or ligand binding domain of the ligand to be "trapped", thereby acting as a mimic to the actual receptor of the molecule.Several ongoing/concluded clinical testing of the combination of TGF-β and PD-1/PD-L1 pathway blocakade in solid tumors.

Fig. 2 .
Fig. 2. 4T-Trap's proposed mechanism of action.A. Activated CD4+ T cells secrete TGF-β1 and induce a suppressive, autocrine TGF-β signaling through TβRII.The activation of TGF-β pathway in the CD4+ T cells prevents them from to efficiently differentiating into T helper type 2 cells (Th2 cells), which leads to the formation of "leaky" tumor vasculatures and tumor growth.B. The F ab regions of 4T-Trap bind the CD4 receptor on T cells while the extracellular domain (ECD) of TβRII on the F c region of 4T-Trap binds to TGF-β ligands in the CD4+ T cell locale.The trapping of TGF-β ligands prevents their binding to the TβRII receptor on the CD4+ T cells.Decreased TGF-β signaling promotes the differentiation of the CD4+ T cells into IL-4 secreting, Th2 T cells.Fully functional Th2 CD4+ T cells then induce the normalization of the tumor vasculature, which leads to tumor cell hypoxia and death.

Fig. 3 .
Fig. 3. Co-targeting of PD-1 and VEGFA may relieve VEGFA-induced T cell exhaustion.A. Tumor and stroma-derived VEGFA binds to VEGFR-2 expressed on activated CD8+ T cells.The binding results in the activation of the master regulator of T cell exhaustion, TOX, which subsequently dials up the expression of multiple immune checkpoints on T cells.TOX activation can also suppress T cell proliferation and cytotoxic activities, resulting in attenuated tumor killing.B. The proposed mechanism of action of a PD1-VEGFA-trap, AK112.Upon binding to the PD-1 receptors expressed on activated CD8+ T cells, AK112 sequester VEGFAS protein in the nearby locale and decreases the activity of VEGFA/ VEGFR-2 pathway signaling in T cells (while minimally impacting the effects of VEGFA signaling elsewhere).Reduced TOX activity relieves CD8+ T cells from exhaustion and dysfunction, which can ultimately lead to increased T cell-induced tumor killing.

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Ther.Author manuscript; available in PMC 2024 March 21.Due low enrollment, the HCC cohort was terminated early.ƗƗ Unlikely to meet its PFS primary endpoint when compared to pembrolizumab, trial is discontinued.See https://www.emdgroup.com/en/news/bintrafusp-alfa-037-update-20-01-2021.htmlƗƗƗ Low accrual and safety concern ^Study was closed after one treatment related death ^^Sponsor decision following information on cases of hyperprogression and early toxicities with bintrafusp alfa in other studies targeting fused with recombinant IL12 cytokine Pharmacol Ther.Author manuscript; available in PMC 2024 March 21.